i H3 
V 1 



-128 



PODBLIGHT OF THE LIMA BEAN CAUSED BY 
DIAPORTHE PHASEOLORUM 



by 



L. L. HARTER 



Reprinted from JOURNAL OF AGRICULTURAL RESEARCH 

Vol. XI, No. 10 : : : : Washington, D. C, December 3, 1917 



A THESIS SUBMITTED TO THE GRADUATE 
FAPULTY CF GEORGE WASHINGTON UNIVERSITY 
FOR THE DEGREE OF DOCTOR OF PHI UOSOPH* 




PUBLISHED BY AUTHORITY OF THB SECRETARY OF AGRICULTURE, WITH THB COOPERATION 
OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS 



WASHINGTON : GOVERNMENT PRINTING OFFICE : 1917 



.B3H3 



PODBLIGHT OF THE LIMA BEAN CAUSED BY 
DIAPORTHE PHASEOLORUM 

By L. h. Harter, 

Pathologist, Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant 
Industry, United States Department of Agriculture 

INTRODUCTION 

The Lima bean {Phaseolus lunatus L.), compared with some of the other 
truck crops, is relatively unimportant from a commercial standpoint. It 
is grown in a small way in the garden by nearly every farmer, and on a 
commercial scale in a few sections of the United States, particularly 
along the Atlantic seaboard and along the Pacific coast. Like many 
other crops grown only for home use or on a commercial scale in a limited 
area, its several pests and diseases have either been overlooked or ignored. 
The Lima bean, however, has its share of diseases, some of which have 
been fairly well studied, while little is known about others. To the 
latter class belongs the podblight, a disease of considerable economic 
importance some seasons in commercial fields. It was first discovered 
by Halsted (16) 1 in 1891, in New Jersey. Further than this nothing is 
known as to the time and place of the origin of the disease. 

There is no evidence from the search of the literature that the disease 
occurs outside of the United States, although it is impossible to state 
definitely that it does not, in view of the great number of species of 
Phoma, Phyllosticta, and closely related genera under which the casual 
fungus may have been described. A careful search through the descrip- 
tions of species of the genera Phoma, Phyllosticta, and Phomopsis on 
different species of Phaseolus has revealed none located in foreign coun- 
tries identical with the organism causing the podblight. It therefore 
appears safe to assume at the present time that the disease is indigenous 
to the United States. 

To judge from published accounts, Halsted (16) was the first to recog- 
nize this disease and found it causing much damage to the pods of pole 
Lima beans in New Jersey in the fall of 1891. He attributed it to a 
species of Phyllosticta, pointing out at the same time that large blotches 
were also produced on the leaves. That he studied the disease with some 
care and did not content himself with a few passing observations is 
evident from the fact that he found that the seeds from infected pods, 
when placed under a suitable environment, such as between moist cloths 
or paper, developed the fruiting bodies of the fungus. 

1 Reference is made by number (italic) to 'i' Lterature cited," p. 302-504. 

Journal of Agricultural Research, Vol. XI, No. 10 

Washington, D. C. Dec. 3, 1917 

kx Key No. G— 128 

(473) 



474 Journal of Agricultural Research vol. xi, No. 10 

In 1892 Ellis and Everhart {15, p. 158) evidently collected the same 
organism on the pods of Phaseolus lunatus at Newfield, N. J., and de- 
scribed it as a new species of Phoma, Phoma subcircinata, pointing out 
that it differed from Phoma leguminum West, in the subcircinate arrange- 
ment of the pycnidia and the rather larger binucleate sporules. Halsted 
later apparently concurs with Ellis and Everhart that this is a new 
species of Phoma, for in 1901 he (27) refers to the disease again by that 
name and enlarges his earlier description. Two illustrations are ap- 
pended to his article, one showing the characteristic appearance of the 
disease on the pods and the other on the leaves, both typical of the trouble 
as we know it at the present time. 

DISTRIBUTION, PREVALENCE, AND LOSS 

For 14 years after this disease was found in New Jersey it had either 
not appeared in any other State or had not been discovered. In 1905, 
however, Clinton (5, p. 265) reports a leafspot of Phaseolus lunatus at 
New Haven, Conn., apparently the same as that described and illustrated 
by Halsted. He states that he did not observe the disease on the pods, 
but adds that it had not been looked for there. Although agreeing 
macroscopically with Halsted's description of the disease on the leaves, 
Clinton entertained some doubt of its identity with Phoma subcircinata 
E. and E. because of the fact that the spores averaged larger (5 to 12 
by 2.5 to 3.5 n) and were occasionally septate. He suggests the pos- 
sibility that the fungus may be Ascochyta phaseolorum Sacc. 

In 1 91 2 Cook (6, p. 51J-518) reports a severe outbreak of a disease of 
pole Lima beans in the State of New Jersey caused by Phyllosticta sp., 
which occurs on leaves and to some extent on the pods and stems. The 
following year it was less severe (7, p. 801), but in 1 914 he (8, p. 472) 
reports the disease again as causing much damage, attributing it to 
Phoma subcircinata E. and E. A leafspot caused by Phyllosticta phaseo- 
lina Sacc. was also commonly met with. 

In view of the relatively few times this disease has been reported 
during a period of 25 years, it is evident that it has generally been either 
overlooked or disregarded. This is probably due to the fact that the 
crop is relatively a minor one and that the disease appears usually after 
the low prices do not justify further pickings. It, however, is much 
more widely distributed than the published reports indicate. It was 
reported (unpublished) to the Plant Disease Survey from one locality 
in North Carolina by R. H. Fulton in 191 3. The writer received and 
studied specimens of badly infected plants from Maryland in 1914 and 
1 91 5, and from Virginia in 191 5. During the seasons of 191 4 and 191 5 
the disease also caused heavy loss to the crop in New Jersey. In con- 
clusion, it may be stated that the disease is rather widely distributed 
along the Atlantic seaboard and causes considerable damage to the crop. 



Gift 
>rgity 
NOV 20 I8ti 



Dec. 3,1917 Podblight of Lima Bean 475 

DESCRIPTION OF PODBLIGHT 

Although the ascogenous stage of this fungus has been connected by the 
writer with the pycnidial stage, the latter is alone concerned in the injury 
to the crop in the field. A macroscopic description of this disease there- 
fore will be confined to the pycnidial or imperfect stage. In the field 
under suitable conditions the disease first appears on the leaves of plants 
from 1 to 2 feet high. Wet weather is conducive to the spread and warm 
weather promotes the development of this as well as many other dis- 
eases of this type. Serious outbreaks have often occurred immediately 
following a few days of rainy, warm weather in fields where previously 
the disease was scarcely noticeable. In such a case the loss is often se- 
rious; but if dry weather follows new growth of leaves and fruit will be 
produced comparatively free of the disease. If, however, the weather 
continues wet, a new outbreak is liable to occur. 

The leaves function as host for the fungus during the earlier part of the 
season, from which it spreads later to the pods. Large, subcircular, 
brown, often bordered patches are produced on the leaves (PI. 42, A). 
These patches, varying in size naturally with age, often attain a diameter 
of 1 to 3 cm. Infection is not restricted to any portion of the leaflets 
but the regions bordering the midrib are most frequently attacked. The 
fungus spreads more or less in all directions from the point of infection, 
but may often be delimited by a large vein or midrib. In this moribund 
tissue, or in tissue soon after it is killed, the fruiting bodies (pycnidia) of 
the fungus break through the epidermis. They are arranged more or 
less concentrically, and appear first as gray or grayish raised pimples, 
which later darken and become nearly black. They therefore may be 
seen as conspicuous, minute black specks on the upper surface of the 
leaves standing apart, sometimes confluent, on a brown background. 
The dead tissues finally become dry and fall out, leaving a ragged hole. 

This disease must not be confused with a leafspot of Lima beans and 
other varieties of beans, as well as of cowpeas, caused by Phyllosticta 
phaseolina Sacc, the spots of which are smaller, more nearly round, and 
the pycnidia smaller and fewer in number. 

The disease appears on the pods usually the latter part of July or early 
in August, at a time when the vines have nearly reached their full devel- 
opment. Under field conditions it is not certain at what stage in the de- 
velopment of the pod infection takes place. Young pods are rarely 
found diseased, and inoculation experiments have shown that they are 
infected less easily than ones nearly matured. The disease progresses 
rather slowly, requiring a week or 10 days after inoculation to produce a 
spot of any size or for the production of pycnidia. The growth of a bean 
pod is comparatively rapid, and it is likely that infection may take place 
when the pods are young, the fungus becoming visible as the pod ap- 
proaches maturity. Infection occurs at any point on the surface of the 



476 Journal of Agricultural Research vol. xi, no. «/ 

pod, but more frequently at or near the ventral suture (PI. 42, B). If 
infection occurs near or at the dorsal or ventral suture, the fungus spreads 
in all directions in a circular or semicircular manner, the hyphae growing 
among the cells of the pod below the epidermis. The extent of the hyphal 
growth of the fungus among the cells is indicated by the darkening of the 
pod above the invaded area, evidently the first signs of the exhaustion of 
the food supply and approaching death of that part of the pod. This is 
soon followed by numerous, minute elevations over the surface, the points 
where the pycnidia are forming. These lifted points are at first gray, but 
as soon as the pycnidia break through the epidermis they become nearly 
black and protrude in a domelike manner from the surface. They are 
often, though not always, arranged concentrically and stand apart ; or two 
or more may be confluent in a cluster or chainlike manner. Upon the 
death of the pod the fungus grows rapidly through the tissues, the 
pycnidia breaking out more or less over the entire surface (PI. 42, C). 
The fruiting bodies which are formed subsequent to the death of the pod 
are usually not formed concentrically. 

Under field conditions the fruiting bodies of the fungus are often found 
on the dead and more rarely on moribund stems. It is exceedingly 
uncommon to find typical infections of living stems, and the writer has 
never been able to produce infection of living stems artificially. The 
pycnidia, however, appear abundantly after the death of the vine on 
stems that have been sprayed with spores of the fungus. 

The disease is much more common and destructive on the pole Lima 
beans than on the bush Limas. 

ETIOLOGY 

The podblight of Phaseolus lunatus is caused by a fungus pathogen to 
which various names have been given at different times. The use of 
different names is largely due to the lack of a knowledge of the life his- 
tory of the fungus and to the practice of many mycologists in separating 
the genera Phyllosticta and Phoma, respectively, according to whether 
the fungus occurs on the leaf, or on the stem, or on other parts of the 
host. 

Halstead (16) first reported this disease in 1891 as causing considerable 
damage to the pods and leaves, and attributed it to a species of Phyllo- 
sticta. He was evidently in doubt as to the species, for he does not 
refer it to Phyllosticta phaseolina Sacc. (30, p. 149), which had been 
described some years earlier and which occurs commonly on Lima beans, 
on Phaseolus diver sifolius, Kansas (Kellerman), New Jersey (Ellis), 
Canada (Dearaess) on Phaseolus perennis, Missouri (Galloway) and on 
cowpea, Kansas (D. B. Swingle). In 1905 Smith (33) proved the patho- 
genicity of this organism on the leaves of Lima beans and other varieties, 
such as kidney and wax, and on cowpeas. He found that the pods 
were not affected. 



Dec. 3. 1917 Podblight of Lima Bean 477 

That the writer has been working with the same fungus which Ellis 
and Bverhart described as Phoma subcircinata there is little doubt. 
Material, collected by Dr. J. B. Ellis at Newfield, N. J., and identi- 
fied by him as Phoma subcircinata, has been examined and found 
to be identical with material collected at Vineland, N. J., only 4 
miles distant from Newfield. It should be mentioned in this connec- 
tion that the material from which Ellis and Everhart made their de- 
scription was likewise collected at Newfield, in 1892. In 1890, three 
years before Ellis and Everhart's description of Phoma subcircinata 
appeared (15, p. 158), Ellis collected and identified as Phyllosticta 
phaseolina some material from Newfield which upon examination the 
writer has found to be identical with material later identified by him 
(Ellis) as Phoma subcircinata and also identical with the material the 
writer has been studying. 

Phoma subcircinata E. and E. was described by Ellis and Everhart 
(15, p. 158) as follows: 

On pods of Lima Beans, Newfield, New Jersey, October, 1892. 

Perithecia subcuticular, 70-90 m diam., sublenticular, subconfluent pierced above, 
membranous, black, subcircinately arranged in large (1 cm.), round, faintly zonate 
spots, finally spreading and occupying the entire surface of the pods. Sporules 
oblong-elliptical, hyaline, 2-nucleate, 5-6 by 2-2.5 M. on simple basidia rather larger 
than the sporules. 

This description fits perfectly the fungus the writer has been studying, 
with the exception of the size of spores and pycnidia. An examination 
of the material collected and identified by Ellis as Phoma subcircinata 
shows the spores to be somewhat larger (6.4 to 8.0 by 2.4 to 3.6 ix, average 
7.4 by 2.95 fj.), and the pycnidia considerably larger (142 to 276 /*, average 
185.8 fi). Material collected and identified by Ellis three years earlier as 
Phyllosticta phaseolina and which he probably later took for Phoma sub- 
circinata, bore spores (6.0 to 8.0 by 2.8 to 3.2 /*, average 7.2 by 2.88 /*) 
and pycnidia (197.5 to 260.0 /*, average 219.0 tx) of about the same size. 
On material which the writer has collected and from which isolations 
have been made for inoculation work the spores varied from 6.0 to 8.6 
by 2.4 to 4.1 v, average 7.50 by 3.23 /x, and the pycnidia from 158.0 to 
475.0 n, average 245.86 fi. From the data at hand it appears evident 
that the fungus described by Ellis and Everhart as Phoma subcircinata 
is the same as the one the writer has been studying. 

In view of the fact that the fungus causing podblight has been con- 
nected with a perfect stage, the genus to which the imperfect form should 
properly belong is of no great consequence, but it may be of interest to 
know that this fungus, like a number of others which have been classed 
as Phoma, belongs to the form genus Phomopsis. In morphological 
structures the podblight fungus is identical with similar structures of the 
genus Phomopsis as laid down in Diedicke's (13) revision of the group — 



478 Journal of Agricultural Research VoLxi,No. 10 

that is, chambering of the pycnidia, formation of sclerotial stroma, 
presence of pycnospores and stylospores, and structure of the pycnidial 
wall. No stroma is formed by this fungus on the leaves. If this fact 
should be disregarded and Saccardo's classification followed, it should b& 
classed as a Dothiorella or a Fusicoccum. Stylospores have been found to 
be abundant in most of the material collected and studied, but none were 
found in the herbarium material collected and identified by Ellis as 
Phoma subcircinata. However, isolations have been made from material 
which did not bear stylospores but developed them in cultures later, 
which shows that, although not always present, the fungus may pro- 
duce them. On the other hand, the organism isolated from specimens 
bearing stylospores do not always produce them in culture. This fun- 
gus is very similar to a large group of other organisms which have long 
been classed as Phoma. In a taxonomic study of the group Diedicke 
(13) transferred a number of species of Phoma to the genus Phomopsis. 
The podblight fungus does not differ essentially morphologically from 
the conidial stage of Diaporthe batatatis (E. and H.) Harter and Field, 
which was first described as Phoma batatae but found later by Harter and 
Field (21) to be a species of Phomopsis. It is similar also to the organism 
causing fruitrot, leafspot, and stemblight of eggplant, which was known as 
Phoma solani on the stem and fruit and as Phyllosticta hortorum on 
the leaves, both of which were found by Harter (20) to be caused 
by the same organism and to belong to the form genus Phomopsis. 

Some years before Phoma subcircinata was described by Ellis and Ever- 
hart, Cooke and Ellis (0, p. 95) described Sphaeria (Diaporthe) phaseo- 
larum occurring on bean stalks as follows: 

Gregaria, tecta. Peritheciis globosis, immersis, minimis. Ostiolis spinaeformibus, 
atris, erumpentibus, Ascis clavatis. Sporidiis lanceolatis, quadrinucleatis. Spo- 
ridia 0.016 mm. 

In the original description no type location was given; neither is it 
apparent in this or subsequent descriptions whether the fungus was found 
on Lima beans or other varieties. In the absence of these facts, together 
with the imperfect description, it would be difficult to identify correctly 
the fungus if it had not been more fully described later by Ellis and 
Everhart (14, p. 460) as follows : 

Diaporthe phaseolorum (C. & E.). 
Sphaeria phaseolorum, C. & E. Grev. VI, p. 93. 
Diaporthe phaseolorum, Sacc. Syll. 2635, Cke. Syn. 2423. 
Exsicc. Ell. N. A. F. 188. 

Perithecia gregarious, buried, very small. Ostiola spine-like, slender, projecting 
for %-yi. mm. Asci clavate, 30-35 by 6-7 /*• Sporidia biseriate, oblong-lanceolate, 
4-nucleate, scarcely or only slightly constricted, 10-12 by 3 m (16 /x long, Cke.). 

On decaying bean vines left exposed through the winter, Newheld, N. J. Mostly 
around the nodes of the stem, the surface mostly blackened and the stroma limited 
within by a black line. 



Dec. 3, i 9 i 7 Podblight of Lima Bean 479 

The writer collected a quantity of Lima beans (pods and stems) at 
Vineland, N. J., in October, 1914, and January, 1915, which bore an 
abundance of pycnidia of the podblight fungus, generally known as 
Phoma subcircinata E. and E. After isolating the causal fungus the ma- 
terial was wintered out at Washington, D. C, and covered with leaves and 
dirt. On June 2 perithecia of the genus Diaporthe were found to be 
abundant on stems and pods of this material. The perithecia were 
scattered and buried in the tissue of the host except the beaks which 
were long and projecting and somewhat bent as shown by Plate 43, D. 
A pycnidial fungus was isolated from single ascospores which was iden- 
tical with the one isolated from the material before it was wintered out 
Subcultures of this strain were used in inoculation experiments and pro- 
duced infections identical with that produced by subcultures from iso- 
lations of single pycnospores. This strain (known as 598), as well as 
others isolated directly from pycnospores, produced stylospores. The 
perithecial stage has never developed in culture. 

The perithecia produced on the wintered-out material varied in size 
from 158.0 to 355.5 fj., average 251.9 n; the asci 28.0 to 46.2 p. by 5.2 to 
8.0 (x, average 37.4 by 6.73 ju; the ascospores 6.4 to 12.0 n by 2.3 to 4.0 n, 
average 9.5 by 2.93 /jl. The perithecia of type material varied in size 
from 158.0 to 237.0 /u, average 215.6 n; the asci 28.0 to 44.0 it by 4.8 to 
8.0 /x, average 33.6 by 7.0 li; the ascospores 8.0 to 12.0 by 2.4 to 4.0 ju, 
average 10.0 by 3.3 ju. From the fact that the ascospores, asci, and peri- 
thecia together with other morphological characters of the two organisms 
are practically identical, it is concluded that they are the same fungus and 
should be known as Diaporthe phaseolorum (C. and E.) Sacc. 

MORPHOLOGY 

PYCNIDIAL STAGE 

From the point of infection the hyphae grow in all directions, invading 
practically all classes of cells except the bast, a layer of which (fig. 1, 
A, B, d) is found just below the epidermis. Figure 1, A and B, c, shows 
parenchyma cells traversed by the hyphae. Soon after the death of the 
cells the pycnidia begin to form, and develop very rapidly thereafter. 
The hyphal growth increases in the cavities below the stomata or in the 
intercellular spaces. From here it passes up between the bast cells which 
are often somewhat separated beneath the stomata. As growth increases, 
gnarls of hyphae accumulate under the stomata and between the 
epidermis and the bast cells (fig. 1, A, B, b). From this point the fungus 
spreads out just beneath the epidermis and develops a more or less 
circular plate, the base of which rests on the bast cells. Figure 1 , A, shows 
an early stage in the development of the pycnidial plate. As the fungus 
growth continues, the epidermis is gradually elevated (fig. 1, B, a) as the 



480 



Journal of Agricultural Research 



Vol. XI, No, io 



result of the pressure below. Definite cell layers of the pycnidium are 
laid down about this time or a little earlier. The pycnidium completes 
its full development soon thereafter (PI. 43, B). 

The inner layer of the pycnidium is composed of hyalin cells from 
which the conidiophores arise. A slight cavity is formed at the center 
concomitant with the release of pressure by the rupture of the epidermis. 
Inclosed in this cavity numerous conidiophores are developed, extending 
toward the center from which the spores are cut off. As the spores 
increase in quantity, the cavity enlarges by the pushing back of the cells 
lining the cavity and probably by further lifting of the upper part of the 
pycnidium. It has not been possible to determine definitely when the 
opening from the pycnidial cavity to the outside through the beak is 
formed, but it probably takes place only after the pressure within has been 
greatly increased by the continued production of spores. It is believed, 




Fig. 1. — Diaporthe phaseolorum: A, An early stage in the formation of a pycnidium. The epidermis (a) 
is not yet disturbed but the hyphae (6) have accumulated directly beneath it. Note the coursing of 
the hyphae through the parenchyma cells (c) . X130. B, A more advanced stage of figure A. Note 
the bulging of the epidermis (a). X 130. 

however, that their expulsion is largely brought about through the ab- 
sorption of water by the gelatinous mass in which the spores are em- 
bedded, a condition which has been found by Stewart (54) to occur for 
Guignardia aesculi (Pk.) Stewart. 

The writer does not wish to leave the impression that the pycnidia 
develop only in the stomatal chambers, but that this is a common and 
natural place for their formation there can be no doubt. Even in young 
pycnidia it is not always easy, and sometimes is impossible, to tell 
whether or not they were being. developed just beneath a stoma. The 
bast cells of the pods of the Lima bean are very thick walled and are 
generally pressed so close together that it would be difficult, if not impos- 
sible, for the fungus to pass between them. These cells, however, are 
separated at certain places, and often just beneath a stoma, so it would 
be logical that they should form in such a cavity. From these facts it is 
evident that, in many cases at least, the pycnidium is developed beneath 
a stoma, and also at such other places where the bast cells are separated. 



Dec. 3, 191 7 



Podblight of Lima Bean 



481 



-G 




Fig. 2. — Diaporthe phas- 
eolorum: Long, slender, 
fragile sporophores with 
two pycnospores at- 
tached, (a) Sporo- 
phores; (6) pycno- 
spores. X500. 



The pycnidia vary greatly in size (158.0 to 475 fi, average 245.86 n 
from host). They are irregular in shape, chambered with an inner 
hyalin layer from which the conidiophores arise, and a dark outer layer 
much thickened and darkened above (PI. 43, B, C), all characters typical 
of the genus Phomopsis. Sometimes the contiguous sidewalls of two or 
more pycnidia are ruptured, forming a single cavity (Pi. 43, E). On the 
other hand, they may be pressed together but separated by a single dark 
wall and their respective hyalin inner layers, thereby 
forming a chain of pycnidia (PI. 43, C). The pyc- 
nidia can hardly be said to be embedded in a true 
stroma. 

The pycnidia on the leaves and stems are lenticu- 
lar in shape, rarely, or not at all chambered (PI. 43, 
A), fewer in number, and smaller than on the pods 
and usually isolated. They vary in size from 197.5 to 
260.0 /x, average 219.0 //. 

The pycnospores are borne on slender, hyalin, sim- 
ple conidiophores (fig. 2, a), i}4 to 3 times the length 
of the pycnospore. They are mostly oblong to fusoid 
(fig. 2, b; 3), hyalin, 1 -celled, usually straight, seldom slightly curved, 
with two large oil droplets. These droplets, sometimes pressed to- 
gether in the center of the spore, give the appearance of a septum, a 
characteristic common to the genus Phomopsis and so deceptive that 
at a casual glance one might believe that a true septum was laid down. 
That the spore is but 1 -celled can readily be demonstrated by clearing 
it with a little salicylic acid. The variation in the size of 
the pycnospores from various sources is seen from the 
following measurements : 

From host. — 6.0 to 8.6 by 2.4 to 4.1 /x, average 7.5 by 
3-23 M- 

Stems of Melilotus alba. — 5.6 to 10.0 by 2.4 to 4.0 /*, 
average 7.82 by 3.1 1 ix. 

Corn meal. — 5.4 to 8.2 by 2.4 to 3.4 //, average 6.49 by 
2.79 p. 

Irish potato cylinders. — 5.4 to 8.5 by 1.7 to 3.0 n, aver- 
age 6.36 by 2.59 (j.. 
Corn-meal agar. — 5.1 to 8.3 by 2.4 to 3.4 /x, average 6.78by 2.85 /x. 
Rice. — 5.4 to 8.5 by 2.0 to 3.4 n, average 6.45 by 2.80 /jl. 
It will be noted that the spores are relatively large in artificial cul- 
tures on stems of Melilotus alba, and small on cooked Irish potato cyl- 
inders. This difference has been found rather constant, and is prob- 
ably associated with vigor of growth. Of all media tried stems of 
M. alba were found exceedingly favorable for spore formation, while 
cooked Irish potato cylinders were rather unfavorable. 



<* %V 

Fig. 3.— Diapor- 
the phaseolo- 
rum: A group of 
pycnospores. 
Note the oil 
droplets and 
variations i n 
size and shape. 
X500. 



482 



Journal of Agricultural Research 



Vol. XI, No. 10 




Under favorable conditions, such as in a nutrient broth or beef agar, 
the pycnospores germinate in about six to eight hours at room temper- 
ature. Previous to germination the spores 
become almost spherical and slightly en- 
larged. Usually but one germ tube develops, 
but occasionally two (fig. 4). One or both 
of the oil globules may disappear at this 
stage or remain until the germ tube is several 
times the length of the spore. Growth is 
rather slow at first, the hyphal growth attain- 
ing a length compared with the diameter 
of the spores as shown in figure 4 in 48 
hours. During this time branching may oc- 
cur, and septa rarely are formed till later. 

The bodies called stylospores are frequently 
found either alone or accompanying the 
Fig. A.—Diaporthe phaseoiorum: Ger- pycnospores in pycnidia on the pods. They 

minating pycnospores. X500. have never been fpund Qn the leayes> 

They are long, slender, hyalin, nonseptate, straight or curved bodies 
(fig. 5) borne on short, rather stout awl- 
shaped simple hyalin stylosporophores (fig. 
6). Figure 5 shows some stylosporo- 
phores with stylospores attached. They 
are produced but sparingly the early part 
of the season or when the pycnospores 
are most abundant. As the season advances 
the stylospores increase in number and 
appear to be associated, though not always, 
with a saprophytic existence. Following 
the death of the host and the gradual 
disappearance of the pycnospores the stylo- 
spores increase and finally become very 
numerous. Specimens which bore practically no stylospores in the fall 

bore them in increasing numbers throughout 
the winter. There is a possibility from the 
data at hand that the stylospores may be 
associated with a lack of food supply. They 
are produced in some culture media freely, 
but not until after the food supply becomes 
more or less exhausted. Although in cultures 
fig. 6-Diaporihe phaseoiorum: a the pycnospores are usually exuded from the 

group of stylosporophores. X500. , , . 

beak in seven days, the stylospores have never 
appeared sooner than 1 1 and generally not before 15 to 30 days after inocu- 
lation. The average size of the winter crop of stylospores developed 




Fig. 5. — Diaporthe phaseoiorum: Styl- 
osporophores. (a) The stylosporo- 
phore is attached to the stylospore. 
(6) Stylospore not attached to the 
stylosporophore. X 500. 




Dec. 3, 1917 



Podblight of Lima Bean 



483 



under natural conditions is considerably larger than those produced in the 
fall or in culture. Measurements made of the fall stylospores from the 
host vary from 11.7 to 31.0 lx by 1.4 to 2.0 fx, average 22.83 by 1.73 M , 
while those collected on February 7 varied from 20.6 to 54.4 /x by 1.38 
to 2.4 ix, average 32.44 by 2.0 ll. They are produced in culture on rice, 
corn meal, Irish potato cylinders, and stems of Melilotus alba and vary 
in length from 11.7 to 54.4 li 
and in width from 1.38 to 
2.4 n. 

The function of these bodies 
is not known. Repeated at- 
tempts have been made to 
germinate them but without 
success. Reddick (29) has 
suggested the possibility that 
they were paraphyses and 
Saccardo (31, p. 264) regards 
them as conidiophores. Von 
Hohnel (23, p. 526), described 
a new genus, Myxolibertiella, 
to include those species hav- 
ing stylospores and pycno- 
spores. He first placed the 
genus in the order Melanco- 
niales but later in the order 
Phomatales. Bubak (2) and 
Diedicke (13) both regard 
them as spores. The latter 

investigator for Convenience FlG- l-—Diaporthe phaseolorum: Mature hyphae. Note the 
, . 1 1 t->. ... septations, oil droplets, and granules. X500. 

designated the Phoma-hke 

spores by the Greek letter alpha (a) and the stylospores by beta (13). 
Shear {32), like Bubak, Diedicke, and others, regards these bodies as 
spores and designates them as scolecospores. 

There is nothing peculiarly distinctive about the mycelium. It is 
hyalin, frequently branched, and septate. It varies considerably in 
width and is often supplied with enlargements which bear a slight re- 
semblance to chlamydospores. When young the cells are supplied with 
small protoplasmic granules. When the hyphae are old, the cells are 
filled more or less with oil globules or droplets (fig. 7) . 




ASCOGENOUS STAGE 



Specimens of Lima beans consisting of stems and pods collected in 
October, 1914, and January 1, 1915, at Vineland, N. J., were wintered 
out at Washington, D. C, and covered over with dirt and leaves. These 



484 Journal of Agricultural Research vo1.xi.Ng. 10 

specimens were kept under observation through the remainder of the 
winter and spring months. Pycnidia and pycnospores were found from 
time to time, as well as an increasing number of stylospores. On June 2 
pods brought into the laboratory bore a considerable number of mature 
perithecia of an ascomycete, which was identified as Diaporthe phaseo- 
lorum (C. and E.) Sacc. The writer was away for five weeks previous to 
June 2 so it is not certain just when the development of perithecia began, 
and for that reason it was not possible to follow the complete course of 
development. The perithecia are scattered and buried beneath the 
surface, most of the beak protruding. Plate 43, D and F, shows dia- 
grammatically the arrangement of the perithecia and relatively the 
depth they are sunken below the surface. Whether or not the perithecia 
are all formed in the cavities of old pycnidia has not been definitely de- 
termined. The pycnidia are rather numerous 
and often coalesce, and as a matter of chance a 
perithecium might preempt a pycnidium and be 
formed therein. It is believed, however, that 
this is not the rule. The perithecia are not irregu- 
lar in shape, as might be expected if they were 
formed in old pycnidia; but they usually bear a 
rather definite and constant form (PI. 43, F). 
They also have separate and distinct walls which 
Fig. s.-Diaporthe pkaseoio- differ f rom the i ayers f the pycnidium. In no 

rum: (a) Asci and (6) asco- 

spores. X500. case have the spores or stylospores ever been 

found associated with a perithecium, although 
these bodies were present on the host at the time. 

The perithecia are slightly flattened, 158 to 355.5 m, average 251.9 n 
in width and from 1 10.6 to 205.4 /*> average 166.5 M m depth. The beaks, 
many times longer than wide, are usually curved or hooked (PI. 43, D), 
rarely straight, tapering gradually to the end. The perithecium, circum- 
scribed by a dark layer of cells, is lined within by a thin, hyalin layer 
from which the asci arise. The perithecium is evidently almost or 
completely matured before the beak is formed. Plate 43, D, shows 
beaks just appearing through the epidermis. Sections through peri- 
thecia with beaks in the incipient stage show the wall layers to be per- 
fectly formed and many mature asci present. The asci (fig. 8, a) are 
sessile, clavate or fusoid, 28.0 to 46.2 /x by 5.2 to 8.0 /z, numerous, 
and contain eight spores arranged mostly biserially. The ascospores 
are apparently bound together or embedded in a gelatinous mass, since 
they are firmly held together even after the apparent disappearance of 
the ascus wall. The ascospores, (fig. 8, b) measuring 6.4 to 12.0 by 2.3 
to 4.0/i, are spindle-shaped, oblong-lanceolate, i-septate at or near the 
center, slightly or not at all constricted, and contain two Ko four oil 
droplets. 




Dec. 3,1917 Podblight of Lima Bean 485 

The ascospores germinate readily in most any nutrient medium in 
about six to eight hours by the development of a single germ tube from 
one or both cells (fig. 9). The hyphae branch is 12 to 24 hours, and septa 
are laid down at about the same time. Growth is very rapid. At ordi- 
nary room temperature a colony from a single spore becomes visible 
to the naked eye in about five days. The ascogenous stage has not been 
produced in culture. The transfer of a colony from a single ascospore 
resulted after 7 to 10 days in the production of the pyenidial stage on 
the same culture media on which pyenidia were developed by a transfer 
of pyenospores in the same length of time. In other words, there is 
no difference between the growth of the ascogenous strains and pyenidial 
strains on the same culture media. 

INOCULATION EXPERIMENTS 

The inoculation experiments on which the conclusions of the parasitism 
of the organism have been drawn were made in the greenhouse. It was 
found that Lima beans grow normally and produce fruit as well under 
greenhouse conditions as in the open field. Opportunities 
were therefore afforded to study the parasitism of this dis- 
ease under controllable conditions and at any time of the 
year most suited to the needs of the work. 

Controls were held with each experiment, the number 
and results of which are shown in Table I following a dis- fig. 9 .—Diap r- 
cussion of the inoculation experiments. The pods unless lum.^Gemt 
otherwise stated were inoculated by inserting spores and nating asco 
hyphse into a wound made by a sterile needle. While 
later evidence will show that under suitable conditions infection may 
take place without wounding, the relative humidity of a greenhouse is 
usually so low that spores smeared on the surface often fail to germi- 
nate. It was necessary, therefore, to protect the spores from drying out 
by inserting them under the epidermis or by other means. In most 
cases it was impossible to protect the spores against drying, owing to 
the fact that when a Lima bean plant is fruiting abundantly it is gener- 
ally too large to confine in an infection cage, though this was done with 
some smaller plants (see p. 486). 

All inoculations were made from cultures grown on stems of Melilotus 
alba. The age of the culture was not considered or found important 
further than that the culture should be vigorous and fruiting abundantly. 
Spores were exuded from the pyenidium in about seven days after the 
culture was inoculated and continued to do so for 30 to 40 days there- 
after or until the culture was dried. Spores were well suited for inocu- 
lation purposes until the culture medium was dried out. 

Inoculations were made with six different strains. By a strain is here 
meant a difference in the source and not a difference in morphology or 




486 Journal of Agricultural Research voi.xi,No. 10 

parasitology, since all were, found to be identical organisms. For the 
sake of convenience in presentation each of the strains was given a sepa- 
rate number as follows: Strain 286 was isolated on October 26, 1914, 
from a Lima bean pod collected from a small patch at Vineland, N. J. 
Stylospores and pycnospores were abundant. Strain 420 was isolated 
from a Lima bean pod collected during the fall of 1914 by Mr. M. B. 
Waite in Maryland. Stylospores, as well as pycnospores, were present 
in abundance. Strain 447 was isolated from material collected on 
January 1, 1915, from the same field as No. 286. Strain 567 is a reisola- 
tion made on April 12, 191 5, from a pod that had been inoculated in the 
greenhouse on March 13, 191 5, with strain 447. Pycnospores were 
abundant, but there were no stylospores. Strain 598, as previously 
stated, resulted from the isolation on June 4, 191 5, of a single ascospore 
from a pod that had been wintered out at Washington, D. C. Strain 
601 was isolated on August 7, 191 5, from leaves of Lima beans collected 
at Cape May, N. J., on August 5, 191 5. 

Other isolations of the same organism not used for inoculation pur- 
poses have also been made for the purpose of identification and com- 
parison. Frequent reisolations were made from inoculated pods, one 
strain (567) of which was used to make inoculations. From specimens 
inoculated with this strain the causal fungus was again recovered, thus 
fulfilling the requirement of Koch's postulates, claimed necessary to 
prove parasitism. 

The first infection experiments were made on January 15, 191 5, when 
20 nearly mature pods on six small plants were inoculated, 10 with 
organism 286 and 10 with organism 420. After the pods were inoculated 
by inserting spores and hyphse, the plants were sprayed with spores 
suspended in water. They were then kept in a paper-covered infection 
cage for 24 hours. On February 20, pycnidia were abundant on the 
stems and on six pods inoculated with No. 286. Of those inoculated 
with No. 420, five pods were dead, and pycnidia were already on four 
and developed on one more after 10 days in a moist chamber. In this 
case there were no leaf infections, though the stems, particularly the 
lower parts, were covered with pycnidia, the pycnidia developing most 
abundantly on the moribund stems and pods. 

On March 13, 21 small plants were sprayed with organism 447. There 
were only three small, immature pods in all and they were inoculated by 
inserting spores and hyphae. These plants were inclosed for 48 hours 
in paper-covered infection cages. Twelve days later the three pods were 
drying up, and pycnidia were present on one and developed on the two 
others after 7 days in a moist chamber. Here again no leaves were 
infected, and the pods did not show typical infections. These results 
were disappointing at the time, but it was discovered later that young 
leaves and pods were difficult to infect, mature parts being much more 
susceptible to the disease. 



Dec. 3,i9i7 Podblight of Lima Bean 487 

On April 9, 1 91 5, twelve almost mature pods were inoculated by 
inserting spores and hyphae in the usual way with organism 447. The 
plants were too large to place in the infection cage. Seven days later 
the tissue for half an inch or so around the point of infection was visibly 
darker. Three days later some of the pods were withering, and pycnidia 
began breaking through the epidermis % to % inch from the point of 
inoculation. As long as the pods remain firm, the pycnidia form in 
more or less concentric rings ; but concomitant with the withering of the 
pods the fungus rapidly invades all the tissue, and pycnidia break through 
the epidermis indiscriminately in a manner shown by Plate 42, C. Eight 
of these pods finally became infected. 

April 20, nine half-grown pods were sprayed with a suspension of 
organism 447 in sterile water and kept in an infection cage for 24 hours. 
These specimens were kept under observation until June 2, but no 
infection had taken place. It is believed that the failure here is again 
due to the relative immaturity of the pods. These results agree with 
those of Pool and McKay (27), who were able to infect only mature 
leaves of sugar beets with Phoma betae (Oud.) Fr. Apparently the writer 
has succeeded in infecting young pods by wounding, but there is reason 
for doubt, owing to the fact that a small wound frequently results in 
serious injury and death of the pod, in which case the fungus quickly 
invades it as a saprophyte. On April 23, spores of organism 447 were 
inserted in five pods, and by June 2 pycnidia and spores were abundant 
on three and a few days later on one more, one remaining sound. 

On June 2, pods bearing perithecia which contained mature asci were 
brought into the laboratory and thoroughly washed and disinfected in 
mercuric chlorid. The perithecia were carefully picked out and macer- 
ated in sterile water in a watch glass. Twenty-one nearly full-grown 
pods were inoculated by inserting ascospores and bits of broken tissue 
into wounds. Twelve days later thirteen pods showed unmistakable 
evidence of the disease, and by June 2 1 all but four of the pods were well 
covered with the fruiting bodies. The writer does not claim any proof 
of the connection of the conidial and perfect stage from this experiment. 
It is included here because it forms a link in the chain of experiments 
performed with this organism. The results are justly open to the criti- 
cism that spores and hyphae of the imperfect stage may have been 
inserted, and it is not unlikely that such was the case. 

On June 3, 191 5, fifteen pods were inoculated in the usual way with 
organism 567. By June 14, pycnidia were abundantly produced over 
an area an inch or more in diameter on ten pods, and on four others 
by June 21. One pod alone remained sound. 

On June 14, seventeen pods were inoculated with organism 598 
(ascospore strain). In four days the tissue around the wound on some 
pods began to darken, showing unmistakable and characteristic symptoms 
of the disease. As the pods were killed by the fungus, they were gathered 



4 88 



Journal of Agricultural Research 



Vol. XI, No. 10 



and all but one bore numerous pycnidia by August 9. Cultures from some 
of these infected pods produced a growth characteristic of the pycnidial 
stage. On the same date spores of organism 598 were smeared on the 
surface of five nearly full-grown pods. There were no signs of infection 
on June 2 1 , but by July 7 three showed evidence of infection, two remained 
sound. Seven pods of various ages were sprayed with organism 598 and 
confined in an infection cage for 30 hours. Pycnidia appeared on one of 
these pods on June 21 and on two others on August 9 as well as on the 
stems and some of the dead branches. The writer was away for sometime 
previous to August 2, and it is not definitely known just how the disease 
developed. It is probable, in view of the fact that the pods were dead 
and studded all over with pycnidia, that they succumbed sometime 
earlier. 

On June 21 twenty-one pods were inoculated with organism 598 in the 
usual way. One diseased pod was removed on June 29, eighteen on July 
7, and one on August 17. One of the inoculated pods could not be found, 
and was therefore regarded as not infected. There were ten checks held 
for this experiment, one of which became infected and the only one of all 
the checks (66) held at various times. 

On August 17, ten pods were inoculated with organism 601, and by 
September 3 six were well covered with pycnidia. Four remained sound. 
Of 19 pods inoculated with 601 on September 7, ten showed typical in- 
fection on September 25. Nine remained sound. 

A summary of the results is given in Table I. 

Table I. — Summary of inoculation experiments of Lima beans 



Organism No. 



Date of in- 
oculation. 



How inoculated. 



Number in- 
oculated. 



Num- 
ber in- 
fected. 



Num- 
ber of 
checks. 



Num- 
ber of 
checks 

in- 
fected. 



286. 

420. 
447- 



447 

447 

447 ;■ 

Fragments of peri- 
thecia contain- 
ing asci from 
pods. 

567 

598 

598 



598. 
598. 
601. 
601. 



1915- 
Jan. 15 



..do 

Mar. 13 



Apr. 9 

Apr. 20 

Apr. 23 

June 2 



June 3 
June 14 
..do 



..do 

June 2 1 
Aug. 17 
Sept. 7 



By insertion of 
spores in a wound. 

do 

Sprayed 

Wounding 

By wounding 

Spraying 

By wounding 

do 



....do 

....do 

Spores smeared on 
pods. 

Sprayed 

By wounding , 

....do 

...do 



10 pods. . . 



..do.... 

21 plants 
3 pods. . 
12 pods. 
9 pods. . 
5 pods.. 
21 pods. 



15 pods. 
17 pods. 
5 pods. . 



7 pods . . . 
21 pods. . 
10 pods . . 
19 pods. . 



4 
17 



14 

16 

3 

3 

20 
6 

10 



1 
/ 

I 



Dec. 3. 1917 Podblight of Lima Bean 489 

PERPETUATION OF THE FUNGUS 

The finding of mature asci of this fungus on June 2, 191 5, and on June 
11, 19 1 6, and later on the dead vines and pods wintered out at Washing- 
ton, D. C, and on similar material under field conditions at Vineland, N. J., 
at about the same season of the year shows one way the fungus may live 
through the winter. This method of perpetuation is, however, not the 
only one. At intervals from October until June pycnospores were fre- 
quently found, and the fungus was isolated at will from field material. 
It is interesting to note in this connection that the pycnospores were 
produced periodically during the winter season, depending upon weather 
conditions. Following a period of a few days of relatively warm, rainy 
weather, they could nearly always be found in more or less abundance. 
For example, pycnospores were found on material examined on October 
24, January 2, 14, and 21, February 7, March 6, April 14, and June 2. 
Isolations were made on October 24, January 2, February 7, May 31, and 
June 2. Similar results were obtained with Phomopsis vexans (Sacc. and 
Syd.) Harter, the pycnospores of which were found in May and June on 
wintered-out material. However, the same material examined in Decem- 
ber, January, February, and April bore no pycnospores but the stylospores 
appeared to be more numerous during the spring months. The appear- 
ance of pycnospores was roughly coordinated with rainfall and tempera- 
ture, since on May 22, following a period of rainy weather and relatively 
high temperatures, they were found in great abundance. 

It is likely also that the organism may live for a considerable time in 
the seed. The writer has isolated it in pure culture from the interior of 
both seeds and pods after surface disinfection in mercuric chlorid. From 
these results it is apparent that the fungus invades all parts and it is not 
unlikely that it is carried fron one season to the next on the seed. That 
the fungus is not readily killed by drying is evident from the fact that it 
was isolated from dried specimens kept in the laboratory for nine months. 
It would probably survive such conditions even longer. These results 
are supported by the earlier work of Halstead (17), who in 1901 called 
attention to the fact that the seeds were invaded by the fungus and sug- 
gested the possibility that it was carried through the season by that 

means. 

MODE OF INFECTION 

It is not always easy to explain how a parasite gains entrance into its 
host. It is safe to conclude that, if a fungus is parasitic, it may enter 
through wounds, it being merely necessary to show that the host is 
subject to wounding under natural conditions. Other types of penetra- 
tion as, for example, through the unbroken epidermis or by way of the 
stomata are not always easy to demonstrate. There is no doubt that 
physical factors such as rainfall, temperature, humidity, and stomatal 
movement all play an important r61e in the penetration of the epidermis 



49° Journal of Agricultural Research vol. xi, No. w 

or stomata by certain parasites. In this connection it is interesting to 
note the results of Pool and McKay (28), who have studied in some 
detail the relation of stomatal movement to infection with Cercospora 
beticola Sacc. These investigators showed that the germ tubes entered 
only when the stomata were open, the movement of which was closely 
associated with a relatively high humidity, warm temperature, and 
light. Their results further showed that mature leaves were more readily 
infected and possessed a pore opening almost twice as large as young 
immature ones. The writer obtained similar results with the Lima bean 
fungus. It was found, as already pointed out, that young immature 
pods were not infected without wounding while mature pods could 
easily be. This difference may be due at least in part to the difference 
in the size of the stomatal aperture but more likely to the fact that im- 
mature stomata do not open, thus preventing the entrance of the hyphae. 
Under humid conditions the spores germinate and produce a germ tube 





r^ 



Fig. 10. — Diaporlhe pkaseolorum: Some of the germ tubes of the pycnospores penetrating the stomata two 
days after the pods were sprayed, and other germinated spores lying alongside the stomata and growing 
toward or away from the aperture. X 260. 

20 or 30 times the length of the spore in 24 to 48 hours. This is rather 
a feeble growth but the spores do not germinate readily in water, and 
then only a small percentage at that. On pods that had been sprayed 
with a spore suspension in water the germ tubes were found penetrating 
the stomata as shown by figure 10. 

It is evidently a matter of chance when a germ tube finds its way into a 
stoma, since, if there was any attractive force, the germ tubes might be 
expected to head that way, which is apparently not the case. The germ 
tubes, as often observed by the writer and illustrated by figure 10, grow 
just as frequently alongside or away from the stomata as toward them, 
showing that there is no appreciable chemotactic influence. Entrance of 
the stoma would be easy so far as relative size of germ tube and stom- 
atal aperature are concerned. In fact, it would not be impossible and 
it may actually happen that the spores fall directly through the pore 
into the intercellular space beneath. Measurements of the pores showed 
them to vary in length from 22 to 28 fx and in width from 5 to 7 /jl when 
open, the germ tube of the spore in width from 2 to 3.5 /*, and the spore 
itself from 6 to 8.6 n by 2.4 to 4.1 fi. Considering the relative size of 



Dec. 3, 1917 



Podblight of Lima Bean 49 l 



the stomatal pore and the spore of the fungus, it is quite reasonable to 
conceive of an infection even during dry times, an event which actually 
does sometimes take place. It must of course be admitted, and the ob- 
served fact supports such a conclusion, that the number of infections by 
a fungus of this type during dry weather is relatively small compared 
with those during a period of rainfall or damp weather. Jones, Giddings, 
and Lutman (23) pointed out that moist, damp weather was conducive 
to the germination of the spores of Phytophthora infestans and made the 
interesting observation that the germ tube penetrated the leaf of an 
Irish potato through the epidermis as well as through the stomata. 
Similar observations were made by Jones (24), who found that the germ 
tube of Macrosporium solani E. and M. may penetrate the epidermis, as 
well as the stomata of Irish potatoes. The writer found no instance of 
penetration of Lima bean pods except through the stomata, and Pool 
and McKay (28) make a similar statement for Cercospora beticola. 

The inoculation experiments already described have shown that 
infection takes place readily through wounds, perhaps made chiefly 
by wind, and by means of pickers. In ordinary farm practice the pole 
Lima bean is usually trained up several feet above the ground. Exposed 
thus to the wind, the vines are whipped around and the pods wounded 
by rubbing against the stems, poles, or wires on which they are trained. 
A careful examination will readily show the bruised spots on pods ex- 
posed to such conditions. Pickers also, in searching through the foliage, 
may likewise brush or press the immature pods against stems or poles, 
bruising them in a similar manner. The reddish, streaked appearance 
in localized spots often observed on bean pods is characteristic of wounds 
made by constant brushing or rubbing of the pod against a hard surface. 
It is in such areas that infections are frequently found. 
The relation of insects to infection has not been studied. 

DISSEMINATION 

The dissemination of the podblight of Lima beans must be considered 
from two standpoints: (1) Over short distances, and (2) over long dis- 
tances, as from one district or State to another. As to the first of these, 
there are several ways in which the spores may be carried from plant 
to plant or from one field to another. It is not unlikely that insects, 
whether they feed on the pods or not, play an important part in the 
distribution of the spores. During wet periods the spores are exuded 
from the pycnidium, and any insect frequenting the diseased pods is 
likely to carry the spores about on its body. Pickers, too, in searching 
through the foliage for mature fruit, are likely also to distribute the 
spores to unpicked pods, where, under favorable conditions, new infec- 
tions will start. It is interesting to note in this connection that this 
and certain diseases of other crops seldom become serious until about 
picking time. There are two possible explanations for this. One is 



49 2 Journal of Agricultural Research voi.xi.no. » 

that the host may be more susceptible at this age; and the other that 
picking of the fruit, especially if the foliage is wet with dew or from 
rain, results in a liberal distribution of the spores. 

That strong winds may be responsible for severe outbreaks of this 
and other diseases is evident from the fact that Cook (6, p. 517-518) 
found a serious outbreak in a certain section of New Jersey previously 
visited by a severe windstorm. It is, of course, natural to conclude, 
as Cook assumed and the writer has frequently observed, that injury 
to pods and foliage during such a storm would add to the chances of 
infection. It is believed that the part played by the wind in dissemi- 
nating fungus spores has been very greatly underestimated. It is a 
well-known fact that the pollen of pine trees is carried long distances. 
Dr. Albert Mann, of the Bureau of Plant Industry, found * a large 
variety of fungus spores collected on a gelatin plate exposed for about 
15 minutes at a distance of 1 mile above the surface of the earth. This 
readily shows that spores are freely air-borne over long distances, and 
this fact alone may account for the serious outbreak of a disease in 
isolated sections. 

Owing to the fact that the causal organism can readily be isolated 
from the seed, it is likely that this affords the principal means of dis- 
tributing it over long distances. Fruiting bodies have also been found 
on infected seed. This shows the great necessity of saving seed only 
from fields where this disease does not occur. 

CULTURAL CHARACTERISTICS OF THE FUNGUS 

The fungus produces hyphse on nearly all of the culture media in 
ordinary use. The pycnidia, on the other hand, are rarely produced 
on agars, but freely on media containing starch, such as rice or corn 
meal. A study of the growth and production of fruiting bodies was 
made on the following media: String-bean agar, Irish potato agar, beef 
agar, corn-meal agar, cooked rice, cooked corn meal, potato cylinders, 
and stems of Melilotus alba. These media were selected because they 
are in common use in the laboratory of Cotton, Truck, and Forage Crop 
disease investigations, and because some (corn meal and rice) were 
known to be particularly favorable for the growth of other very similar 
organisms, thus affording an opportunity for comparison. Other media 
might have served the same purpose. 

The experiment was carried out by inoculating five tubes (100 c. c. 
flasks, in case of corn meal) with spores from a 10-day-old culture on 
stems of Melilotus alba. After inoculation the tubes were kept in the 
light and exposed to the temperature of the laboratory room (2i°-25°C). 

String-bean agar. — This on the whole has been a poor medium for this fungus. 
Growth of hyphae started promptly and was visible in two days in slanted tubes of 

1 Unpublished data. 



Dec. 3, 1917 Podblight of Lima Bean 493 

the medium. At the end of 9 days three dark bodies, which were at that time thought 
to be p}'cnidia, and which probably were sterile ones, were developed. These cul- 
tures were kept under observation for 72 days, and no more pycnidia and no spores 
were found. 

Irish potato agar. — There was a visible vegetative growth in two days which 
continued to increase for about one week. A few scattered pycnidia appeared in all 
tubes but one. These were kept under observation for 72 days, and no spores or 
stylospores were ever produced. 

BEEP agar. — A rapid vegetative growth during the first seven days of growth. 
Thereafter the hyphae became fluffy and cottony. No pycnidia were formed in any 
of the tubes during the whole course of the experiment. 

Corn-meal agar. — Growth was not visible in two days, but in four days the 
hyphae had spread over an area J4 inch in diameter. In six days dark specks appeared 
which later developed into mature pycnidia. At the end of 13 days the slimy white, 
creamy masses began to exude from the pycnidia. These cultures were frequently 
examined thereafter for stylospores, but none were found until after 34 days, when 
they appeared in great abundance. When stylospores and pycnospores are both 
produced, the former always appear after, and never before, the production of the 
latter. 

Cooked rice. — Rice has proved to be a good medium for the growth of this organ- 
ism. Hyphae were conspicuous and pycnidia began to form in 4 days. From that 
time the pycnidia increased in number and spores began to exude in 7 days. A 
heavy, black stromatic mass, in which the pycnidia were embedded, was formed. 
As the medium dried out, these stromatic masses were left as conspicuous domelike 
projections over the surface of the medium. The pycnospores were exuded in slimy, 
yellowish, viscid masses in such quantities as to flow over the top of the stroma and 
mostly to cover the surface of the medium. Stylospores were found sparingly in 22 
days and abundantly in 34 days. 

Cooked corn meal. — Corn meal was found to be one of the best media for this and 
other closely related fungi, such as Phomopsis vexans, Plenodomus destruens, and 
Diaporthe batatatis. In 5 days the hyphae covered most of the surface of the corn 
meal in 100-c. c. flasks, and pycnidia began to form. The pycnidia were produced 
in great numbers in a more or less well-developed stroma which bore characteristics 
similar to those on rice. The small slimy droplets containing pycnospores were 
first noticed at the end of 13 days. The slimy exudate increased in amount until 
the droplets became quite large and flowed over the sides of the stroma, eventually 
covering nearly the whole surface of the medium. The stroma and pycnidia were 
dark on top and somewhat raised above the surface of the medium. As the corn meal 
dried out, the surface became more or less crusty and rugose. Stylospores were found 
for the first time at the end of 22 days. 

Potato cylinders. — On this medium abundant mycelium was produced. A 
cottony, white growth covered one-third of the potato slant in two days and com- 
pletely in four days. In five days incipient pycnidia were seen which later developed 
into a more or less stromatic mass. These masses were at first whitish on top, but 
later turned darker. In 22 days pycnospores were exuding slightly, and stylospores 
were present. At the end of 34 days the stylospores had become more abundant than 
on any other medium; the pycnospores, on the other hand, were relatively few. 

Stems oe melilotus alba. — This medium, everything considered, is the best 
of any so far used. It gives a maximum amount of spores with a minimum of mycelium. 
There were no signs of growth in two days, but in five days numerous pycnidia began 
to form, and these soon became conspicuous as black specks studding the surface of 
the stems. The beaks were comparatively long and conspicuous. In one week 
under optimum conditions the viscid, creamy droplets in which the spores are em- 



494 Journal of Agricultural Research vol. xi, No. 10 

bedded were noticeable on the end of the beak. These droplets gradually enlarged 
and finally overflowed the pycnidium, coalescing with similar droplets from other 
pycnidia and thereby forming an almost continuous liquid covering of the substratum. 
A stroma was not formed on stems of Melilotus alba. In this particular experiment 
no stylospores were formed. In other cultures, however, of the same organism on 
M. alba stems a few stylospores were found. 

EFFECT OF LIGHT ON GROWTH 

The literature is full of the records of experiments on the effect of light 
on growth and development of chlorophyll-bearing plants, as well as 
fungi. It would be useless to review all or any considerable part of these 
records here. It is evident, however, from a perusal of some of these 
publications that no general, sweeping conclusions can be drawn that 
will apply to all fungi alike. Recent results, on the other hand, show that 
under similar conditions individual fungi may be expected to respond 
differently. In support of this last statement, reference may be made 
to the results of Coons (10), who found that light was a determining factor 
in the production of pycnidia with Plenodomus fuscomaculans (Sacc.) 
Coons. The writer (19), on the other hand, working with a different 
species of Plenodomus, P. destruens Harter, found that the absence of 
light did not inhibit the production of pycnidia. That the absence of 
light very greatly inhibited, but did not entirely prevent, the production 
of pycnidia by Diaporihe batatatis, was shown by Harter and Field (21) 
in another experiment. The writer has obtained similar results, as yet 
unpublished, with other fungi. 

The influence of light on growth and production of fruiting bodies of 
the pycnidial stage of Diaporihe phaseolorum was determined by inocu- 
lating stems of Melilotus alba in test tubes and exposing one-half of the 
set to light and excluding the light from the other tubes by wrapping 
with black paper, which was found by previous test to prqhibit the 
passage of light. The tubes were all tightly plugged with cotton. The 
cotton plugs alone were left exposed, so as to permit aeration. The two 
series were then placed on a table in the center of the laboratory room 
and kept under observation for 25 days. The cultures began to dry up 
about that time and development was accordingly arrested. The tubes 
kept in the dark were examined for a few minutes every few days for 
the purpose of taking notes and were for that length of time exposed 
to the light. While this brief period can not be regarded as having 
no influence on the results, exposure to the light for such a short time 
would certainly have no marked effect. The growths of mycelium in 
the cultures in the light and those in the dark were about the same at 
the end of three days, but from that time the mycelial growth in 
the dark was relatively more abundant and more cottony. In tubes 
in the light, on the other hand, there was, as has always been, a minimum 
of mycelium on stems of Melilotus alba. At the end of six days pycnidia 



Dec. 3, 1917 



Podblight of Lima Bean 495 



were forming in large numbers in all tnbes in the hght and m smaller 
Zmbers in all those in the dark. The ratio between the number of 
Turn a formed in the light and in the dark remained nearly constant 
the fruiting bodies forming in all tubes, but they were better developed 
n the light In one week spores began to exude from the pycmdia 
both in the light and in the dark. From this time spores continued 
to be exuded, but here again the amount of the exudate was relatively 
less in the dark. Toward the close of the experiment, however, there 
appeared to be a relative increase in the exudate from the pycmdia 
formed in tubes in the dark. But one conclusion can be d™™*"" 
these results, that while the dark retards the production of fruit ng 
bodies and accelerates vegetative growth, it is not as marked as the 
results obtained with other fungi by the writer and others. 

INFLUENCE OF TEMPERATURE ON GROWTH 

To determine the influence of temperature on growth 12 (No 2, 3, 5. 
6 8 q .0 15 17, 18, .9, and 20) of the 20 chambers ,n an Altmann 
incubator were used. Tubes of cooked rice were inoculated with spores 
oTthe nngus and immediately placed in the chambers, where they were 
kept exposed to the respective temperatures for a period of 27 days, at 
thfendTwhich time growth had practically stopped. No growth took 
place in chambers 2 (4.1° C). 3 (6..° C), 19 (35-4° O. and 20 (38 8 O 
and but a scant growth in 18 (32.9° C). The optimum growth was 
ana DU , , 5 , <,on.nHi7(an l, C) The growth of hypha: 
found in chambers 1 5 (26.5° C.) and 17 U0.5 <-.)■ s ^ 

decreased relatively with the increa* and decrease of the temperature 

from these limits of the optimum. In chambers 9 (17.4 C .), 10 (19.4 
from these > v ^ stromatlc f 

pseudoparenchymo^ tissue were produced which macroscopically looked 
hke pyenidia. A careful examination of these bodies, however showed 
hat'while they bore some resemblance to pyenid, 1 ,n< , cavity had b 
produced within them and no pyenospores were found. The cause or 
tto was later found to be due to a lack of sufficient aeration, resulting 
probably in a reduced oxygen supply. A subsequent expenmen, in 
which cultures of the organism on cooked rice were exposed in a chamber 
ta^hfchm^rf the oxygen had been removed by a mixture of pyrogalhc 
acid audllm hvdroxfd showed that while mycelial growth continued 
under such conditions the production of fruiting bodies was inhibited. 
In" add ta o tte f act that'the cultures in the Altmann chambers were 
tent in the dark, the absence of oxygen, which in itself was found as 
aheady Sown, to reduce somewhat the production of «b^». 
evidentlv accounted for the failure to produce pyenidia in the cited cases. 
The growth of the podblight fungus in artificial cultures is schemati- 
cally shown in figure n. 



496 



Journal of Agricultural Research 



Vol. XI, No. 10 



/O /S 2Q PS SO .3 6- 



4P 



INFLUENCE OF TOXIC AGENTS ON GERMINATION OF SPORES 

Perhaps no chemical substance has been more generally studied in its 
relation to the growth of plants than copper salts. This is perhaps due 
to its well-known efficiency as a fungicide, following the discovery of which 
a large amount of work was undertaken to determine the concentration 
of copper salts necessary to prevent germination or bring about the death 
of the spores of a great variety of fungi. Much of this work was done 
with saprophytes, and consequently has little more than a scientific 
interest, while the practical side of the problem was evident from the fact 
that the toxicity to germinating spores of many well-known parasites was 
established. Because of the various uses that chemical substances may 
be put to, such as antiseptics, disinfectants, etc., the toxic action of many 

salts, metals, and acids, 
alone and in combina- 
tion, on flowering 
plants and fungus 
spores have been ex- 
tensively studied, and 
as a result an extensive 
literature on the sub- 
ject has accumulated. 
As might be expected, 
the results have been 
very variable and often 
contradictory. In gen- 
eral, however, all in- 
vestigations have shown that copper salts, among other chemical 
substances, are exceedingly toxic to all germinating spores and to the 
seedlings of flowering plants. Coupin (11) has shown by experiments 
that 1 part of copper sulphate to 700,000,000 parts of water is suffi- 
cient to retard the root growth of wheat seedlings, and the writer has 
proved (18) that a small amount of a single alkali salt, such as 
sodium chlorid, sodium carbonate, and magnesium sulphate in water 
cultures, inhibits the growth of the same plant. Heald (22) found 
1/51,200 and 1/25,600 gram-molecule per liter of copper sulphate to be 
toxic to Pisum sativum and Zea mays, respectively. According to Moore 
and Kellerman (26) 1 part of copper sulphate to 500,000 parts of water 
killed Closterium moniliferum in 4 days and 1 part of copper sulphate to 
3,000,000 parts of water killed Anabaena flos-aqwz in three days. 
Uroglena americana was even more sensitive and was killed in 16 hours 
in 1 part of copper sulphate to 10,000,000 parts of water. 

These few references show the extreme sensitiveness of some of the 
flowering plants and some algse to copper salts, and at the same time the 
variability in their resistance. Although not strictly comparable with 




Fig. i 1 —Diaporthe phaseolorum: Graphic representation of growth on 
cooked rice at different temperatures. 



Dec. 3, 1917 Podblight of Lima Bean 497 

flowering plants, because of the different methods or experimentation 
and standards of measurement, the spores of fungi show equally great 
variability and sensibility to copper salts and other toxic substances. 
Clark (3) found that Penicillium glaucum was injured by copper sulphate 
at a concentration somewhere between N 1 2,048 and N '/ 1,024, while 
Aspergillus flavus would endure a concentration of N/512 to NI256, two 
to four times as great. Crandall (12), on the other hand, showed that 
the apple scab fungus is quite resistant to copper sulphate, the spores 
being only slightly retarded in germination in a solution of 1 to 100,000, 
while a concentration of about 1 to 10,000 was necessary to entirely 
prevent germination. At this latter concentration some of the common 
molds, however, grew quite well. Temperature was found by Brooks 
(1) to influence the percentage of germination of the spores of certain 
fungi in the same concentration of a toxic substance. By an extensive 
series of experiments he found that Penicillium glaucum gave medium 
growth in N/128 copper sulphate at 20 C, Monilia fructigena did not 
germinate in N/16 copper sulphate at 25 to 30 C, but at 15 C. 12 per 
cent, at io° C. 30 per cent, and at 5 C. 49 per cent. 

No attempt has been made to review all the extensive literature on the 
effect of toxic substances on plant growth. The results of the work of 
the different investigators are very conflicting, due, perhaps in part at 
least, to the different methods of experimentation employed and the 
different standards of measurement. 

During the time the podblight fungus of Lima bean has been under 
investigation many interesting observations have been made from time 
to time on the effect of certain chemical substances commonly used in the 
laboratory on the germination of the spores. The use of a solution of 
mercuric chlorid for disinfecting field specimens preparatory to isolating 
the fungus led to a study of the strength of this solution necessary to kill 
the spores ,n a given time. A similar study was made with formaldehyde 
because of its possible use in disinfecting infected seed, and with copper 
sulphate, a well-known constituent of Bordeaux mixture, sometime rec- 
ommended as a spray in controlling the disease in the field. 

METHOD OF EXPERIMENTATION 

Preliminary experiments showed that the use of Van Tieghem cells 
to determine the toxic limit for the germination of the spores was ex- 
ceedingly unreliable and was consequently abandoned. Although this 
method has been generally used, it is likely that many of the conflicting 
results may be attributed to it. In fact, the writer found that each 
test in Van Tieghem cells might be expected to give different results, 
probably owing to the variation in vapor pressure or to some slight 
accident overlooked, unexpected, and unaccounted for. After con- 
sidering the different methods that might be used, the writer concluded 



498 Journal of Agricultural Research voLxi.no. 10 

to employ the method frequently used in medical bacteriology to de- 
termine the germicidal value of certain chemicals. This takes more 
into account the actual death of the organism rather than the strength 
that inhibits growth. It seems that the same method should apply 
equally well to fungi and is easier of determination and more reliable, 
since the length of time the organism is exposed plays an important 
part also, and can be more accurately gauged. It is a well-known fact 
that a fungicide may merely inhibit growth when, the spores are exposed 
to it for a certain length of time but may kill them on prolonged ex- 
posure. In these experiments the time the spores were exposed to the 
fungicide was relatively short, the concentration of the solution being 
proportionately strong to produce death in a comparatively short time. 
This method eliminated the necessity of taking into account, among 
other things, the temperature which Brooks (1) found to play such an 
important part in germination of the spores of some fungi. 

The pycnospores of Diaporthe phaseolorum were found to grow well in 
beef agar + 15 on Fuller's scale; and therefore this medium was used in 
which to test germination. The spores from a culture on Melilotus alba 
were transferred by a small platinum loop to a solution of the fungicide 
of the desired concentration and mixed by a wide circularly swinging 
motion of the arm with the plugged end up, or by vigorous shaking. At 
the end of one minute, two minutes, and every two minutes thereafter 
up to the suspected limit of endurance, a loopful of the mixture was 
transferred to a tube of liquefied agar and, after mixing by shaking, a plate 
was poured. Control plates were made by transferring a loopful of 
spores from the same test culture to a sterile water tube and a loopful 
from this tube to a tube of liquefied agar. This method was followed 
with each of the fungicides and repeated as often as necessary to establish 
the toxic limit. The plates were kept at room temperature in the 
laboratory. The colonies in the control plates appeared in about two 
days. Notes were taken from time to time for seven days, after which 
they were thrown out, for, if growth took place at all, it would do so 
before that time. 

RESULTS OF EXPERIMENTS 

The results of these experiments are given in Table II. 

The mercuric-chlorid and copper-sulphate solutions were made in 
advance and kept corked in the ice box until needed. Because of the 
instability of the formaldehyde, the solutions were prepared just before 
using and in such a way that the fractional parts given in Table II are 
based on a hypothetical formaldehyde solution of a strength of 100 per 
cent. 



Dec. 3, 191 7 



Podblight of Lima Bean 



499 



Table II. — The results of exposing pycnospores of Diaporthe phaseolorum to different 
strengths of mercuric chlorid, copper sulphate, and formaldehyde for different lengths of 
time 



Time of 


Copper sulphate. 


Formaldehyde. 


Mercuric chlorid. 


exposure. 


NI12.5 


NI31.25 


NI62.S 


1:100 


1:250 


1:500 


NI13S 


Nli,3so 


N 12,700 


NI4.050 


Minutes. 
I 
2 

4 


a 100 

35 
5 


100 
65 

55 
25 
15 
10 

5 
2 


100 
90 


75 



85 

30 




100 
100 

85 

10 





O 


O 
O 


5° 
O 


IOO 
IOO 

75 
5° 


6 








8 


75 












10 














12 


45 
















14 
16 
















20 
















18 




















20 






2 





































a In this and corresponding columns are given the percentage of germination as estimated at the close of 
the experiment. Water controls were made in every case in which the germination was arbitrarily placed 
at 100 per cent. These plates, together with those poured from a 1 minute exposure, served as controls for 
comparison in estimating percentages of germination. 

Reference to the table will show that the spores were nearly all killed 
in four minutes in a N/12.5 copper-sulphate solution, and in 14 minutes 
in a N/31.25, and in 20 minutes in a N/62.5 solution. It is interesting 
to compare these results with those of Clark (3), who found that the 
spores of several fungi exposed in a copper-sulphate solution were killed 
or injured as follows: Aspergillus flavus (?) Link N/4 and greatly injured 
in N/256, Sterigmatocystis niger Von Tieghem N/8 and greatly injured in 
N/128, Oedocephalum albidum Sacc. N/64 an d greatly injured at N/256, 
Botrytis vulgaris Fr. greatly injured at N/128, and Penicillium glaucum 
Link N/i and greatly injured at N/256. As will be seen, there is a wide 
difference between the strength of the solution necessary to kill and that 
required to injure greatly the spores. The writer found that none of the 
spores exposed for one minute to a solution of mercuric chlorid at a con- 
centration of N/135 an d N/1,350 would germinate, and that only 50 per 
cent germinated when exposed for the same length of time in a concen- 
tration of N/3,700. At the greater dilution, N/4,050, 50 per cent germi- 
nated after an exposure for six minutes. Clark (3) found that spores of 
A. flavus, S. niger, P. glaucum were killed in a N/ 4,096 mercuric chlorid, 
O. albidum in a N/ 16,384, and B. vulgaris in a N/ 65, 536. The spores of 
these fungi were killed in the solutions of the toxic substance more dilute 
than that required to kill the pycnospores of Diaporthe phaseolorum, but 
they were likewise subjected to the solution for a longer time. In 
another work Clark (4) has shown that Rhizopus nigricans was killed in 
75 seconds in a solution of mercuric chlorid 1 to 3,700. 

Formaldehyde was found to be very toxic to the spores, only 10 per 
cent germinating after a six-minute exposure in a i-to-500 solution, and 



500 Journal of Agricultural Research vol. xi, no. io 

30 per cent after a two-minute exposure in a i-to-250 solution, and 

only 75 per cent in a 1 -to- 100 solution after one minute. Clark (3) 

found that 1 part by weight in 273,066 parts of water permitted the 

germination of but 10 per cent of the spores of Oedocephalum albidum 

after 1 1 hours' exposure, as compared with 95 per cent after four hours 

in the controls, and greatly injured the mycelial development. 

As regards the results, it should be stated that in all cases where the 

spores were not killed the germination was very much retarded. The 

colonies in the controls became visible to the naked eye in about 48 

hours. A long exposure in a weak solution retarded germination to 

about the same^ degree that a short exposure in a correspondingly more 

concentrated solution. That the germination of Lima bean seeds is not 

injured by exposure for 10 minutes in the strongest solutions of mercuric 

chlorid, formaldehyde, and copper sulphate to which the spores were 

subjected is shown by a series of experiments discussed under "Control 

measures." 

CONTROL MEASURES 

Halsted (17) was the first, and probably the only one, who has ever 
done any experimental work on the control of the podblight of Lima 
beans. In these experiments spraying the dwarf Limas with Bordeaux 
mixture or soda-Bordeaux mixture yielded very favorable results. 
Although he evidently did not spray the pole Limas, he suggests that the 
disease could be checked by so doing. It is now known that the fungus 
attacks the leaves before the pods are set, and it is likely the pods become 
infected from them. In view of this fact, the first spraying should be done 
when the plants are 1 or 2 feet high, and should be repeated often enough 
to keep the foliage covered. 

From badly infected pods the fungus invades the seed, where it may 
live for a considerable time. Infected seed are usually dark, shriveled, 
and immature. They probably would not grow, and, if they did, would 
give weak, sickly plants. Only plump, bright seed should be planted. 
Sound seed may be disinfected to kill the adhering spores by submerging 
for 5 or 10 minutes in a solution of mercuric chlorid (1 : 1,000), formalde- 
hyde (1:100), or copper sulphate (1:100), after which they should be 
rinsed in water. 

A series of experiments has shown that germination was not injured by 
such a treatment. Healthy Lima bean seeds were sorted out; a part 
were treated for 5 minutes and a part for 10 minutes. After rinsing in 
sterile water, they were laid between moistened filter paper and ger- 
minated. The results showed that seeds exposed for 10 minutes gave 
just as good germination as those soaked in sterile water for the same 
length of time. It should be mentioned in this connection that those 
soaked in sterile water were all more or less covered with saprophytic 
molds, while those treated with mercuric chlorid, formaldehyde, and 
copper sulphate were comparativelv free. 



Dec. 3 , 1917 Podblight of Lima Bean 501 

SUMMARY 

(1) The podblight of Lima beans is probably indigenous to the United 
States. 

(2) It was first recognized by Halsted in New Jersey in 1891, and has 
since been found in the States of Virginia, Maryland, Connecticut, and 
North Carolina. 

(3) The disease forms circular brown spots on the leaves, and large 
unsightly spots on the nearly mature pods and on the stems. Numerous 
pycnidia are produced in the diseased areas. 

(4) The loss, which in some fields is very large, is confined mostly to 
the pods. 

(5) The Lima bean podblight fungus has been known as Phoma 
subcircinata E. and E. almost since its first discovery, although in a few 
cases it has been referred to as a species of Phyllosticta. 

(6) The writer isolated single ascospores of a Diaporthe found on 
specimens wintered out and made inoculations with pure cultures of 
such strains which produced a disease identical with that produced by 
inoculations with pure cultures of a pycnospore strain. In 1 892 Diaporthe 
phaseolorum was completely described, which agrees with the ascogenous 
fungus the writer has been studying. The fungus causing the disease 
is therefore referred to as Diaporthe phaseolorum (C. and E.) Sacc. 

(7) The hyphae course through and among the parenchymous cells 
of the pod, forming pycnidia just under the epidermis at points where 
the bast cells are more or less separated. 

(8) Stylospores are abundantly found on field material and readily 
produced in culture on some media. The pycnospore stage belongs to 
the form genus Phomopsis and not to Phoma. 

(9) The disease is readily produced by artificial inoculation with spores 
from both the pycnidial and ascogenous strains. 

(10) Wounding of the pod is not necessary for infection, as the germ 
tubes may enter through the stomata. 

(11) The fungus is carried through from one season to the next, either 
by periodic production of pycnospores and the persistence of hyphae or 
by the production of an ascospore stage. 

(12) The disease may be carried on the seed and spread by such 
agencies as wind, by the process of picking, and possibly by means of 
insects. 

(13) The fungus fruits well on stems of Melilotus alba, on rice, corn 
meal, and other starch media, and but poorly or not at all on the agars 
tried with the exception of corn-meal agar. 

(14) Cultures in the dark fruited more slowly than those in the light 
and had increased vegetative growth. 

(15) No growth takes place at a temperature below 6.i° nor higher 
than 35. 4 C. during a period of 27 days. 



502 Journal of Agricultural Research vol. xi. No. 10 

(16) Dilute solutions of formaldehyde, copper sulphate, and mercuric 
chlorid are toxic to the spores. The germination of the spores is greatly 
retarded when exposed to a solution too dilute to kill them in a given 
time. 

(17) To control the podblight it is recommended that clean seed be 
selected. They should then be disinfected in mercuric chlorid, formalin, 
or copper sulphate. The plants should be sprayed in the field the first 
time when they are 1 to 2 feet tall, and often enough thereafter to keep 
the foliage covered. 

LITERATURE CITED. 

(1) Brooks, Charles. 

1906. temperature and toxic action. In Bot. Gaz., v. 42, no. 5, p. 359-375, 
12 fig. Literature cited, p. 375. 

(2) Bubak, Franz, and Kabat, J. E. 

1905. VIERTER BEITRAG ZUR PILZFLORA VON THIOL. In OsteiT. Bot. Ztschr., 

Jahrg. 55, no. 2, p. 73-79. (Continued article.) 

(3) Clark, J. F. 

1899. ON THE TOXIC EFFECT OF DELETERIOUS AGENTS ON THE GERMINATION 
AND DEVELOPMENT OF CERTAIN FILAMENTOUS FUNGI. In Bot. Gaz., 
v. 28, no. 5, p. 289-327; no. 6, p. 378-404, 10 diagr. Bibliography, 
p. 402-404. 
(4) 

19OI. ON THE TOXIC VALUE OF MERCURIC CHLORIDE AND ITS DOUBLE SALTS. In 

Jour. Phys. Chem., v. 5, no. 5, p. 289-316, 7 fig. 

(5) Clinton, G. P. 

1906. REPORT OF THE BOTANIST. NOTES ON FUNGUS DISEASES, ETC., FOR 

1905. In Conn. Agr. Exp. Sta., 29th Ann. Rpt., [i904]/o5, p. 263-277. 

(6) Cook, M. T. 

1913. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 33d 

Ann. Rpt., [i9ii]/i2, p. 509-527. 

(7) 

1914. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 34th 

Ann. Rpt., [i9i2]/i3, p. 793~8i7> 13 fi g- 
(8) 

191 5. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 35th 

Ann. Rpt., [i9i3]/i4, p. 467-476. 
(9) Cooke, M. C, and Ellis, J. B. 

1878. new jersey fungi. In Grevillea, v. 6, no. 39, p. 81-96. (Continued 
article.) 
(10) Coons, G. H. 

1916. FACTORS INVOLVED IN THE GROWTH AND THE PYCNIDIUM FORMATION OF 

plEnodomus fuscomaculans. In Jour. Agr. Research, v. 5, no. 16, 
p. 713-769. Literature cited, p. 766-769. 
(n) Coupin, H. 

19OI. SUR LA SENSIBIEIT6 DES v£g6TAUX SUPERIEURS A DES DOSES TRES 
FAIBLES DE substances ToxiquES. In Compt. Rend. Acad. Sci. 
[Paris], t. 132, no. 10, p. 645-647. 

(12) Crandall, C. S. 

1909. bordeaux mixture. 111. Agr. Exp. Sta. Bui. 135, p. 199-296, 8 fig. 

(13) Diedicke, H. 

1911. die gattung phomopsis. In Ann. Mycol., Jahrg. 9, no. 1, p. 8-35, 3 pi. 



Dec. 3. 191 7 Podblight of Lima Bean 503 

14) Ellis, J. B., and Everhart, B. M. 

1 892. THE NORTH AMERICAN PYRENOMYCETES. 793 p., 41 pi. Newfield, N. J. 

15) 

1894. NEW SPECIES OF NORTH AMERICAN FUNGI FROM VARIOUS LOCALITIES. 

In Proc. Acad. Nat. Sci. Phila., 1893, p. 128-172. 
16) Halsted, B. D. 

1892. lima bean diseases. In N. J. Agr. Exp. Sta., 12th Ann. Rpt, 1891, 
p. 287. 

17) 

1901. bean diseases and THEIR remedies. In N. J. Agr. Exp. Sta. Bui. 151, 
28 p., 9 fig., 4 pi. 
18) Harter, L. L. 

1905. THE VARIABHJTY OF WHEAT VARIETIES IN RESISTANCE TO TOXIC SALTS. 

U. S. Dept. Agr. Bur. Plant Indus. Bui. 79, 48 p. Bibliography, 
p. 47-48. 
19)- 

20) 



1913. the foot-rot OF THE SWEET potato. In Jour. Agr. Research, v. 1, 
no. 3, p. 251-274, 1 fig., pi. 23-28. 



1914. FRUIT-ROT, LEAF-SPOT, AND STEM BLIGHT OF THE EGG PLANT CAUSED BY 

phomopsis vexans. In Jour. Agr. Research, v. 2, no. 5, p. 331-338, 
1 fig., pi. 26-30. Literature cited, p. 338. 
21) and Field, Ethel C. 

1913. A DRY ROT OF SWEET POTATOES CAUSED BY DIAPORTHE BATATATIS. 

U. S. Dept. Agr. Bur. Plant Indus. Bui. 281, 38 p., 4 fig., 4 pi. 

22) Heald, F. D. 

1896. ON THE TOXIC EFFECT OF DILUTE SOLUTIONS OF ACIDS AND SALTS UPON 

plants. In Bot. Gaz., v. 22, no. 2, p. 125-153, pi. 7. 

23) Hohnel, Franz von. 

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PLATE 42 
Diaporthe phaseolorum: 

A. — A leaflet of Phaseolus lunatus L. , showing a number of ragged holes of various 
sizes caused by the Lima bean podblight fungus. This specimen was collected at 
Cape May, N. J., August 5, 1915. The disease was produced on pods by inoculation 
with the organism (601) isolated from this leaf. Natural size. 

B. — A green Lima bean pod photographed 10 days after inoculation with the ascos- 
pore strain. Pycnidia are abundantly formed over an area about y 2 inch in diameter; 
outside of this is another somewhat darkened area which has already been invaded 
by the fungus, but the pycnidia have not yet broken through the epidermis. Natural 
size. 

C. — A pod showing the characteristic manner in which the fungus rapidly overruns 
it soon after it is killed, pycnidia forming indiscriminately over the entire surface. 
Natural size. 



Podblight of Lima Bean 



Plate 42 





\ 




Journal of Agricultural Research 



Vol. XI, No. 10 



Podblight of Lima Bean 



A 



Plate 43 








Journal of Agricultural Research 



Vol. XI, No. 10 



PLATE 43 
Diaporthe phaseolorum: 

A. — A vertical section through a pycnidium from the leaf. X 130. 

B. — A vertical section through a pycnidium from a pod showing the beak and the 
thickened dark wall surrounding it. Note also conidiophores arising from hyalin inner 
ayer. Some spores are attached. X130. 

C. — A vertical section through the epidermis and underlying host of a portion of a 
diseased pod showing the connection and chambering of the pycnidia. X65. 

D. — A somewhat diagrammatic drawing of a portion of the surface of a Lima bean 
pod, showing the distribution of the perithecia, the shape of the beaks and their pro- 
jection from the surface. The beaks of two perithecia may be seen just breaking 
through the epidermis. X 16. 

E. — A surface view of a portion of a bean pod showing arrangement of the pycnidia 
and fusion of the beaks. X65. 

F. — A vertical section through a perithecium from a pod. Note the dark wall 
surrounding the perithecium in which the asci are inclosed; also the portion of the 
beak buried below the surface of the pod. The outer end of the beak has been cut 
away. X130. 



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002 810 714 6 f 



